Drosophila Blood-brain Barrier Research Articles

The Drosophila blood–brain barrier invades the nervous system in a GPCR-dependent manner

The blood–brain barrier (BBB) represents a crucial interface between the circulatory system and the brain. In Drosophila melanogaster, the BBB is composed of perineurial and subperineurial glial cells. The perineurial glial cells are small mitotically active cells forming the outermost layer of the nervous system and are engaged in nutrient uptake. The subperineurial glial cells form occluding septate junctions to prevent paracellular diffusion of macromolecules into the nervous system. To address whether the subperineurial glia just form a simple barrier or whether they establish specific contacts with both the perineurial glial cells and inner central nervous system (CNS) cells, we undertook a detailed morphological analysis. Using genetically encoded markers alongside with high-resolution laser scanning confocal microscopy and transmission electron microscopy, we identified thin cell processes extending into the perineurial layer and into the CNS cortex. Interestingly, long cell processes were observed reaching the glia ensheathing the neuropil of the central brain. GFP reconstitution experiments highlighted multiple regions of membrane contacts between subperineurial and ensheathing glia. Furthermore, we identify the G-protein-coupled receptor (GPCR) Moody as negative regulator of the growth of subperineurial cell processes. Loss of moody triggered a massive overgrowth of subperineurial cell processes into the CNS cortex and, moreover, affected the polarized localization of the xenobiotic transporter Mdr65. Finally, we found that GPCR signaling, but not septate junction formation, is responsible for controlling membrane overgrowth. Our findings support the notion that the Drosophila BBB is able to bridge the communication gap between circulation and synaptic regions of the brain by long cell processes.

The Drosophila blood–brain barrier regulates sleep via Moody G protein-coupled receptor signaling

Sleep is vital for most animals, yet its mechanism and function remain unclear. We found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. We observed increased BBB permeability in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, gαo, loco, lachesin, or neuroglian-each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep.

Hormonal gatekeeping via the blood-brain barrier governs caste-specific behavior in ants

Here, we reveal an unanticipated role of the blood-brain barrier (BBB) in regulating complex social behavior in ants. Using scRNA-seq, we find localization in the BBB of a key hormone-degrading enzyme called juvenile hormone esterase (Jhe), and we show that this localization governs the level of juvenile hormone (JH3) entering the brain. Manipulation of the Jhe level reprograms the brain transcriptome between ant castes. Although ant Jhe is retained and functions intracellularly within the BBB, we show that Drosophila Jhe is naturally extracellular. Heterologous expression of ant Jhe into the Drosophila BBB alters behavior in fly to mimic what is seen in ants. Most strikingly, manipulation of Jhe levels in ants reprograms complex behavior between worker castes. Our study thus uncovers a remarkable, potentially conserved role of the BBB serving as a molecular gatekeeper for a neurohormonal pathway that regulates social behavior.

Modulation of sleep by trafficking of lipids through the Drosophila blood-brain barrier.

Endocytosis through Drosophila glia is a significant determinant of sleep amount and occurs preferentially during sleep in glia of the blood-brain barrier (BBB). To identify metabolites whose trafficking is mediated by sleep-dependent endocytosis, we conducted metabolomic analysis of flies that have increased sleep due to a block in glial endocytosis. We report that acylcarnitines, fatty acids conjugated to carnitine to promote their transport, accumulate in heads of these animals. In parallel, to identify transporters and receptors whose loss contributes to the sleep phenotype caused by blocked endocytosis, we screened genes enriched in barrier glia for effects on sleep. We find that knockdown of lipid transporters LRP1&2 or of carnitine transporters ORCT1&2 increases sleep. In support of the idea that the block in endocytosis affects trafficking through specific transporters, knockdown of LRP or ORCT transporters also increases acylcarnitines in heads. We propose that lipid species, such as acylcarnitines, are trafficked through the BBB via sleep-dependent endocytosis, and their accumulation reflects an increased need for sleep.

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship.

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.

The nuclear receptor Hr46/Hr3 is required in the blood brain barrier of mature males for courtship.

The blood brain barrier (BBB) forms a stringent barrier that protects the brain from components in the circulation that could interfere with neuronal function. At the same time, the BBB enables selective transport of critical nutrients and other chemicals to the brain. Beyond these functions, another recently recognized function is even less characterized, specifically the role of the BBB in modulating behavior by affecting neuronal function in a sex-dependent manner. Notably, signaling in the adult Drosophila BBB is required for normal male courtship behavior. Courtship regulation also relies on male-specific molecules in the BBB. Our previous studies have demonstrated that adult feminization of these cells in males significantly lowered courtship. Here, we conducted microarray analysis of BBB cells isolated from males and females. Findings revealed that these cells contain male- and female-enriched transcripts, respectively. Among these transcripts, nuclear receptor Hr46/Hr3 was identified as a male-enriched BBB transcript. Hr46/Hr3 is best known for its essential roles in the ecdysone response during development and metamorphosis. In this study, we demonstrate that Hr46/Hr3 is specifically required in the BBB cells for courtship behavior in mature males. The protein is localized in the nuclei of sub-perineurial glial cells (SPG), indicating that it might act as a transcriptional regulator. These data provide a catalogue of sexually dimorphic BBB transcripts and demonstrate a physiological adult role for the nuclear receptor Hr46/Hr3 in the regulation of male courtship, a novel function that is independent of its developmental role.

The Fly Blood-Brain Barrier Fights Against Nutritional Stress.

In the wild, animals face different challenges including multiple events of food scarcity. How they overcome these conditions is essential for survival. Thus, adaptation mechanisms evolved to allow the development and survival of an organism during nutrient restriction periods. Given the high energy demand of the nervous system, the molecular mechanisms of adaptation to malnutrition are of great relevance to fuel the brain. The blood-brain barrier (BBB) is the interface between the central nervous system (CNS) and the circulatory system. The BBB mediates the transport of macromolecules in and out of the CNS, and therefore, it can buffer changes in nutrient availability. In this review, we collect the current evidence using the fruit fly, Drosophila melanogaster, as a model of the role of the BBB in the adaptation to starvation. We discuss the role of the Drosophila BBB during nutrient deprivation as a potential sensor for circulating nutrients, and transient nutrient storage as a regulator of the CNS neurogenic niche.

Glial ER and GAP junction mediated Ca2+ waves are crucial to maintain normal brain excitability.

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca2+ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca2+ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca2+ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca2+ signaling. Here we identify a role for ER store-operated Ca2+ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. We show that PG cells display diverse Ca2+ activity that varies based on their locale within the brain. Ca2+ signaling in PG cells does not require extracellular Ca2+ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca2+ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation.

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.

The Serine Protease Homolog, Scarface, Is Sensitive to Nutrient Availability and Modulates the Development of the Drosophila Blood-Brain Barrier.

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as “brain sparing.” However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood–brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood–brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood–brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB.

Plasticity of Carbohydrate Transport at the Blood-Brain Barrier.

Neuronal function is highly energy demanding, requiring efficient transport of nutrients into the central nervous system (CNS). Simultaneously the brain must be protected from the influx of unwanted solutes. Most of the energy is supplied from dietary sugars, delivered from circulation via the blood-brain barrier (BBB). Therefore, selective transporters are required to shuttle metabolites into the nervous system where they can be utilized. The Drosophila BBB is formed by perineural and subperineurial glial cells, which effectively separate the brain from the surrounding hemolymph, maintaining a constant microenvironment. We identified two previously unknown BBB transporters, MFS3 (Major Facilitator Superfamily Transporter 3), located in the perineurial glial cells, and Pippin, found in both the perineurial and subperineurial glial cells. Both transporters facilitate uptake of circulating trehalose and glucose into the BBB-forming glial cells. RNA interference-mediated knockdown of these transporters leads to pupal lethality. However, null mutants reach adulthood, although they do show reduced lifespan and activity. Here, we report that both carbohydrate transport efficiency and resulting lethality found upon loss of MFS3 or Pippin are rescued via compensatory upregulation of Tret1-1, another BBB carbohydrate transporter, in Mfs3 and pippin null mutants, while RNAi-mediated knockdown is not compensated for. This means that the compensatory mechanisms in place upon mRNA degradation following RNA interference can be vastly different from those resulting from a null mutation.

A Role for the Drosophila Blood-Brain Barrier in the Regulation of Sleep

Sleep is vital for most animals, yet its mechanism and function remain unclear. The blood-brain barrier GPCR moody is upregulated in sleep-deficient insomniac (inc) null Drosophila, suggesting a link between the blood-brain barrier (BBB) and sleep. We found that permeability of the BBB–the organ required for maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain–displays circadian clock-dependent rhythmicity that is aligned with sleep/wake cycles. We observed increased BBB permeability in the chronically sleep-deprived sleep mutants insomniac, wakeD1, wakeD2 and sleepless, as well as acutely sleep deprived animals. In addition to tracers, sleep deprivation-induced BBB changes also increased penetration of drugs used in the treatment of brain pathologies. After chronic or acute sleep deprivation, rebound sleep, or administration of the sleeping aid gaboxadol, normalized BBB permeability, showing that sleep deprivation effects on the BBB are reversible. Altering BBB permeability alone through glia-specific suppression of moody, lachesin, or gαo, each a well-studied regulator of BBB function, was sufficient to induce robust sleep phenotypes, demonstrating a tight link between BBB permeability and sleep. Together these studies indicate a role for the BBB as a novel regulatory center for sleep.

An original infection model identifies host lipoprotein import as a route for blood-brain barrier crossing

Pathogens able to cross the blood-brain barrier (BBB) induce long-term neurological sequelae and death. Understanding how neurotropic pathogens bypass this strong physiological barrier is a prerequisite to devise therapeutic strategies. Here we propose an innovative model of infection in the developing Drosophila brain, combining whole brain explants with in vivo systemic infection. We find that several mammalian pathogens are able to cross the Drosophila BBB, including Group B Streptococcus (GBS). Amongst GBS surface components, lipoproteins, and in particular the B leucine-rich Blr, are important for BBB crossing and virulence in Drosophila. Further, we identify (V)LDL receptor LpR2, expressed in the BBB, as a host receptor for Blr, allowing GBS translocation through endocytosis. Finally, we show that Blr is required for BBB crossing and pathogenicity in a murine model of infection. Our results demonstrate the potential of Drosophila for studying BBB crossing by pathogens and identify a new mechanism by which pathogens exploit the machinery of host barriers to generate brain infection.

Steroid Hormone Entry into the Brain Requires a Membrane Transporter in Drosophila.

Steroid hormones control various aspects of brain development and behavior in metazoans, but how they enter the central nervous system (CNS) through the blood-brain barrier (BBB) remains poorly understood. It is generally believed that steroid hormones freely diffuse through the plasma membrane of the BBB cells to reach the brain [1], because of the predominant "simple diffusion" model of steroid hormone transport across cell membranes. Recently, however, we challenged the simple diffusion model by showing that a Drosophila organic anion-transporting polypeptide (OATP), which we named Ecdysone Importer (EcI), is required for cellular uptake of the primary insect steroid hormone ecdysone [2]. As ecdysone is first secreted into the hemolymph before reaching the CNS [3], our finding raised the question of how ecdysone enters the CNS through the BBB to exert its diverse role in Drosophila braindevelopment. Here, we demonstrate in the Drosophila BBB that EcI is indispensable for ecdysone entry into the CNS to facilitate brain development. EcI is highly expressed in surface glial cells that form the BBB, and EcI knockdown in the BBB suppresses ecdysone signaling within the CNS and blocks ecdysone-mediated neuronal events during development. In an exvivo culture system, the CNS requires EcI in the BBB to incorporate ecdysone from the culture medium. Our results suggest a transporter-mediated mechanism of steroid hormone entry into the CNS, which may provide important implications in controlling brain development and behavior by regulating steroid hormone permeability across the BBB.

Drosophila SMN2 minigene reporter model identifies moxifloxacin as a candidate therapy for SMA.

Spinal muscular atrophy is a rare and fatal neuromuscular disorder caused by the loss of alpha motor neurons. The affected individuals have mutated the ubiquitously expressed SMN1 gene resulting in the loss or reduction in the survival motor neuron (SMN) protein levels. However, an almost identical paralog exists in humans: SMN2. Pharmacological activation of SMN2 exon 7 inclusion by small molecules or modified antisense oligonucleotides is a valid approach to treat SMA. Here we describe an in vivo SMN2 minigene reporter system in Drosophila motor neurons that serves as a cost-effective, feasible, and stringent primary screening model for identifying chemicals capable of crossing the conserved Drosophila blood-brain barrier and modulating exon 7 inclusion. The model was used for the screening of 1100 drugs from the Prestwick Chemical Library, resulting in 2.45% hit rate. The most promising candidate drugs were validated in patient-derived fibroblasts where they proved to increase SMN protein levels. Among them, moxifloxacin modulated SMN2 splicing by promoting exon 7 inclusion. The recovery of SMN protein levels was confirmed by increased colocalization of nuclear gems with Cajal Bodies. Thus, a Drosophila-based drug screen allowed the discovery of an FDA-approved small molecule with the potential to become a novel therapy for SMA.

Degradation of Extracellular Matrix by Matrix Metalloproteinase 2 Is Essential for the Establishment of the Blood-Brain Barrier in Drosophila

SummaryThe blood-brain barrier (BBB) is an essential system that isolates the central nervous system from the internal environment. Increasing evidence has begun to reveal the molecules that are required for BBB integrity. However, how these components are regulated remains unclear. Here we report that a matrix metalloproteinase, Mmp2, is essential for the establishment of the BBB in Drosophila. In the absence of mmp2, the BBB becomes leaky, which allows the tracer to penetrate the brain. Moreover, the expression pattern of a junctional component, Neuroglian, is altered. We also find that the regulation of the amounts of particular extracellular matrix components is critical for BBB establishment. Furthermore, the process of mesenchymal-epithelial transition of BBB-forming cells is perturbed in the absence of Mmp2. These data indicate that the presence of Mmp(s), which is typically considered to be a risk factor for BBB degradation, is essential for BBB integrity in Drosophila.

Circadian Control of Animal Physiology

We are interested in the mechanisms that generate circadian (~24 hour) cycles of physiology and behavior. Our studies use primarily a Drosophila model, which was invaluable for dissecting molecular mechanisms of the clock, but they also include mammalian models where appropriate. We are interested in the control of behavior by brain clocks and in the control of physiology by brain and peripheral clocks. We recently identified a clock in the Drosophila blood brain barrier (BBB) that confers a 24 hour rhythm on to permeability of the BBB. Specifically, the BBB clock regulates the activity of efflux transporters, such that these transporters pump xenobiotics out of the brain during the day, but are less active at night, thereby increasing BBB permeability at night. The underlying mechanism involves rhythmic expression of gap junctions that serve to maintain 24 hour cycles of magnesium, a positive regulator of efflux transporters, in BBB cells. As many drugs are targets of efflux transporters, we asked if permeability of such drugs is different at different times of day. Indeed, we found that phenytoin/Dilantin has time‐of‐day specific effects in a drosophila seizure model. In recent work, we have characterized rhythms of the BBB in mammalian cells and are addressing the relevant mechanisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Brain and bone cancer targeting by a ferrofluid composed of superparamagnetic iron-oxide/silica/carbon nanoparticles (earthicles)

Despite the advances in molecularly targeted therapies, delivery across the blood-brain barrier (BBB) and the targeting of brain tumors remains a challenge. Like brain, bone is a common site of metastasis and requires therapies capable of discerning the tumor from its healthy cellular milieu. To tackle these challenges, we made a variation on the previously proposed concept of the earthicle and fabricated an aqueous, surfactant-free ferrofluid containing superparamagnetic iron oxide nanoparticles (SPIONs) coated with silicate mesolayers and carbon shells, having 13 nm in size on average. Nanoparticles were synthesized hydrothermally and characterized using a range of spectroscopic, diffractometric, hydrodynamic and electron microscopy techniques. The double coating on SPIONs affected a number of physicochemical and biological properties, including colloidal stability and cancer targeting efficacy. Nanoparticles decreased the viability of glioblastoma and osteosarcoma cells and tumors more than that of their primary and non-transformed analogues. They showed a greater preference for cancer cells because of a higher rate of uptake by these cells and a pronounced adherence to cancer cell membrane. Even in an ultralow alternate magnetic field, nanoparticles generated sufficient heat to cause tumor death. Nanoparticles in MDCK-MDR1 BBB model caused mislocalization of claudin-1 at the tight junctions, underexpression of ZO-1 and no effect on occludin-1 and transepithelial resistance. Nanoparticles were detected in the basolateral compartments and examination of LAMP1 demonstrated that nanoparticles escaped the lysosome, traversed the BBB transcellularly and localized to the optic lobes of the third instar larval brains of Drosophila melanogaster. The passage was noninvasive and caused no adverse systemic effects to the animals. In conclusion, these nanoparticulate ferrofluids preferentially bind to cancer cells and, hence, exhibit a greater toxicity in these cells compared to the primary cells. They are also effective against solid tumors in vitro, can cross the BBB in Drosophila, and are nontoxic based on the developmental studies of flies raised in ferrofluid-infused media. Statement of SignificanceWe demonstrate that a novel, hydrothermally synthesized composite nanoparticle-based ferrofluid is effective in reducing the viability of osteosarcoma and glioblastoma cells in vitro, while having minimal effects on primary cell lines. In 3D tumor spheroids, nanoparticles greatly reduced the metastatic migration of cancer cells, while the tumor viability was reduced compared to the control group by applying magnetic hyperthermia to nanoparticle-treated spheroids. Both in vitro and in vivo models of the blood-brain barrier evidence the ability of nanoparticles to cross the barrier and localize to the brain tissue. These composite nanoparticles show great promise as an anticancer biomaterial for the treatment of different types of cancer and may serve as an alternative or addendum to traditional chemotherapies.

Endocytosis at the Drosophila blood-brain barrier as a function for sleep.

Glia are important modulators of neural activity, yet few studies link glia to sleep regulation. We find that blocking activity of the endocytosis protein, dynamin, in adult Drosophila glia increases sleep and enhances sleep need, manifest as resistance to sleep deprivation. Surface glia comprising the fly equivalent of the blood-brain barrier (BBB) mediate the effect of dynamin on sleep. Blocking dynamin in the surface glia causes ultrastructural changes, albeit without compromising the integrity of the barrier. Supporting a role for endocytic trafficking in sleep, a screen of Rab GTPases identifies sleep-modulating effects of the recycling endosome Rab11 in surface glia. We also find that endocytosis is increased in BBB glia during sleep and reflects sleep need. We propose that endocytic trafficking through the BBB represents a function of sleep.

The Drosophila Blood-Brain Barrier Adapts to Cell Growth by Unfolding of Pre-existing Septate Junctions

The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. Here, we used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. We show that septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish.